Systems and methods of delivery of bioactive agents using bacterial toxin-derived transport sequences

ABSTRACT

The field of the present invention relates, in part, to a strategy for novel pharmaceutical applications. More specifically, the present invention relates to a genetically detoxified form of  Vibrio cholera  exotoxin (cholix) and the use of cholix-derived polypeptide sequences to enhance intestinal delivery of biologically-active therapeutics. Importantly, the systems and methods described herein provide for the following: the ability to deliver macromolecule doses without injections; the ability to deliver cargo, such as (but not limited to) siRNA or antisense molecules into intracellular compartments where their activity is required; and the delivery of nanoparticles and dendrimer-based carriers across biological membranes, which otherwise would have been impeded due to the barrier properties of most such membranes.

RELATED PATENT APPLICATIONS

This application is the 371(c) national phase entry of PCT applicationPCT/US2011/001602 having an international filing date of Sep. 15, 2011,which claims benefit of U.S. Provisional Application No. 61/403,394,filed on Sep. 15, 2010, each incorporated in its entirety by referenceherein.

TECHNICAL FIELD

The field of the present invention relates, in part, to a strategy fornovel pharmaceutical applications. More specifically, the presentinvention relates to a non-toxic mutant form of the Vibrio choleraCholix gene (ntCholix), a variant of Cholix truncated at amino acid A³⁸⁶(Cholix³⁸⁶) and the use of other various Cholix-derived polypeptidesequences to enhance intestinal delivery of biologically-activetherapeutics. Importantly, the systems and methods described hereinprovide for the following: the ability to deliver macromolecule doseswithout injections; the ability to deliver cargo, such as (but notlimited to) siRNA or antisense molecules into intracellular compartmentswhere their activity is required; and the delivery of nanoparticles anddendrimer-based carriers across biological membranes, which otherwisewould have been impeded due to the barrier properties of most suchmembranes.

BACKGROUND ART

The majority of currently-approved small molecule drugs are absorbedacross the mucosa of the small intestine to provide delivery to thesystemic circulation. In fact, small molecule drugs are selected basedupon their stability and efficient absorption across intestinal mucosae.A similar oral delivery of biologically-active polypeptides (referringto a polymer composed of amino acid residues; typically defined as aprotein or peptide) has been a long-standing goal of the pharmaceuticalindustry. As the gastrointestinal (GI) tract is designed to digestdietary proteins and peptides, there are numerous physical,physiological, and biological barriers that limit the feasibility oftherapeutic proteins and peptides uptake from the intestine in a mannersimilar to that achievable with small molecules; Mahato, R. I., et al.,Crit Rev Ther Drug Carrier Syst, 20(2-3):p. 153-214 (2003).

A number of technologies have been identified that can be used toprotect therapeutic proteins and peptides through the stomach, allowingthem to reach the absorptive surface of epithelial cells in the smallintestine and separating them from the gastric and intestinalenvironments that function to destroy dietary proteins and peptides.Unfortunately, however, the efficient transport across this simple,single layer of cells remains a substantial barrier due to theintracellular trafficking to destructive lysosome compartments afterendosomal uptake of polypeptides at the luminal surface; Woodley, J. F.,Crit Rev Ther Drug Carrier Syst, 11(2-3):p. 61-95 (1994). Indeed, thisbarrier is designed to inhibit uptake of proteins and peptides untilthese macromolecules can be sufficiently degraded for absorption throughamino acid and di- or tri-peptide transporters. In this regard, a numberof efforts have been examined to overcome the physical, physiological,and biological barriers of the intestinal mucosae.

There are two basic routes across the simple epithelium that constitutesthe cellular barrier of the intestinal mucosae. Specifically, onceacross the covering mucus layer, a molecule could move between adjacentepithelial cells (paracellular route) or move through cells(transcellular route) via a series of vesicles that traffic within, butdo not mingle, contents with the cytoplasm; T. Jung et al., Eur J PharmBiopharm, 50:147-160 (2000). In other words, in both routes, a transportprotein or peptide therapeutic does not enter into the cell but ratherstays in an environment external to the cell's cytoplasm.

The primary barrier to casual movement of therapeutic protein andpeptide movement through the paracellular route is a complex of proteinsat the apical neck of these cells known as the tight junction (TJ).While transient opening and closing of TJ structures can facilitatetransport of peptides across intestinal epithelia, this approach has keylimitations: e.g., it does not work well for molecules above ˜5 kDa; ithas the potential for non-selective entry of materials into the bodyfrom the intestinal lumen; and it represents a route that involves onlya small fraction of the surface area of the intestinal epithelium.

The primary barrier to casual migration of protein or peptidetherapeutics across cells via the transcellular route is a defaultvesicle trafficking that delivers the contents of these vesicles to adestructive (lysosomal) pathway. As compared to the paracellular route,movement through the vesicular transcellular route can accommodatematerials as large as 100 nm in diameter, involves essentially theentire epithelial cell surface, and can be highly selective in uptake ofmaterials through the use of receptor-ligand interactions for vesicleentry. Thus, the transcellular route is very appealing for theepithelial transport of protein or peptide therapeutics if thedestructive pathway can be avoided.

Some pathogens have solved the trafficking barrier problem, asdemonstrated by the efficient transcytosis of secreted polypeptidevirulence factors which function to facilitate and/or stabilizeinfection of a host. Exotoxins represent a class of proteins released bya variety of microorganisms which function as potent virulence factors.Exotoxins function on multi-cellular organisms with the capacity to actsas potent toxins in man; Roszak, D. B., and Colwell, R. R., MicrobiolRev 51:365-379 (1987). These proteins commonly kill or inactivate hostcells through mechanisms that involve selective disruption of proteinsynthesis. Accordingly, only a few molecules are required to kill,consistent with the observation that bacterial exotoxins are some of themost toxic agents known. A subset of these proteins comprised of thefamily of proteins that consists of diphtheria toxin (DT) fromCorynebacterium diphtheria, exotoxin A from Pseudomonas aeruginosa (PE),and a recently identified protein termed Cholix from Vibrio cholerafunction to intoxicate host cells via the ADP-ribosylation of elongationfactor 2 (EF2); Yates, S. P., et al., Trends Biochem Sci, 31:123-133(2006). These exotoxins are synthesized as a single chain of amino acidsthat fold into distinct domains that have been identified as havingspecific functions in targeting, entry, and intoxication of host cells.

The biology of exotoxin A from Pseudomonas aeruginosa (PE) has recentlybeen described; Mrsny, R. J., et al., Drug Discov Today, 7(4): p. 247-58(2002). PE is composed of a single chain of 613 amino acids having atheoretical molecular weight (MW) of 66828.11 Da, an isoelectric point(pI) of 5.28, and that functionally folds into three discrete domains,denoted domain I (Ala¹-Glu²⁵²), domain II (Gly²⁵³-Asn³⁶⁴), domain III(Gly⁴⁰⁵-Lys⁶¹³, and which contains a ADP-ribosyltransferase activitysite), and a short disulfide-linked loop linking domains II and IIIwhich is known as the Ib loop (Ala³⁶⁵-Gly⁴⁰⁴). The organization of thesedomains at pH 8.0 have determined from crystal diffraction at aresolution of ˜1.5 Å; Wedekind, J. E. et al., J Mol Biol, 314:823-837(2001). Domain I (Ia+Ib) has a core formed from a 13-stranded β-roll,domain II is composed of six α-helices, and domain III has a complexα/β-folded structure. Studies have supported the idea that the modularnature of PE allows for distinct domain functions: domain I binds tohost cell receptors, domain II is involved in membrane translocation,and domain III functions as an ADP-ribosyltransferase. It appears thatPE is secreted by P. aeruginosa in close proximity to the epithelialcell apical surface, possibly in response to environmental cues and/orcellular signals; Deng, Q. and J. T. Barbieri, Annu Rev Microbiol, 62:p.271-88 (2008). Once secreted, internalization into cells occurs afterdomain I of PE binds to the membrane protein α2-macroglobulin, a proteinwhich is also known as the low-density lipoprotein receptor-relatedprotein 1 (LRP1) or CD91; see, e.g., FitzGerald, D. J., et al., J CellBiol, 129(6):p. 1533-41 (1995); Kounnas, M. Z., et al., J Biol Chem,267(18): p. 12420-3 (1992). Following internalization, PE avoidstrafficking to the lysosome and is instead efficiently delivered to thebasolateral surface of the cell where it is released in abiologically-active form; Mrsny, R. J., et al., Drug Discov Today, 7(4):p. 247-58 (2002). Once across the epithelium, PE functions as avirulence factor by entering into CD91-positive cells within thesubmucosal space (macrophage and dendritic cells) where it thenintersects with an unfolding pathway that leads to the cytoplasmicdelivery of domain III; see, e.g., Mattoo, S., Y. M. Lee, and J. E.Dixon, Curr Opin Immunol, 19(4): p. 392-401 (2007); Spooner, R. A., etal., Virol J, 3: p. 26 (2006).

Vibrio cholerae bacterium is best known for its eponymous virulenceagent, cholera toxin (CT), which can cause acute, life-threateningmassive watery diarrhea. CT is a protein complex composed of a single Asubunit organized with a pentamer of B subunits that binds to cellsurface G_(M1) ganglioside structures at the apical surface ofepithelia. CT is secreted by V. cholera following horizontal genetransfer with virulent strains of V. cholerae carrying a variant oflysogenic bacteriophage called CTXf or CTX_(φ). Recent choleraoutbreaks, however, have suggested that strains of some serogroups(non-O1, non-O139) do not express CT but rather use other virulencefactors. Detailed analyses of non-O1, non-O139 environmental andclinical data suggested the presence of a novel putative secretedexotoxin with some similarity to PE.

Jorgensen, R. et al., J Biol Chem, 283(16):10671-10678 (2008) reportedthat some strains of V. cholerae did, in fact, contain a protein toxinhaving similarity to PE and which they termed Cholix toxin (Cholix).Compared to PE, Cholix has a slightly larger theoretical MW (70703.89Da) and a slightly more acidic theoretical pI (5.12). The crystalstructure of the 634 amino acid Cholix protein has been resolved to ˜2Å. The domain structure and organization was found to be somewhatsimilar to PE: domain I (Val¹-Lys²⁶⁵), domain II (Gly²⁶⁶-Ala³⁸⁶), domainIII (Arg⁴²⁶-Lys⁶³⁴), and a Ib loop (Ala³⁸⁷-Asn⁴²⁵). Additionalstructural similarity to PE includes: a furin protease site for cellularactivation; a C-terminal KDEL sequence that can route the toxin to theendoplasmic reticulum of the host cell; and an ADP-ribosyltransferaseactivity site within domain III.

Remarkably, PE and Cholix share no significant genetic and limitedsimilarity by amino acid alignment. Searching the genome of V. cholerafor PE-like nucleotide sequences fails to result in a match of any kind.It is only at the protein sequence level is there the hint that anPE-like protein could be produced by this bacterium. Even here, there isonly a 32% homology between the amino acid sequences of PE and Cholixwith similarities (42% homology) being focused in the ADP ribosylationelements of domain III, and with low levels of amino acid homology(˜15-25%) for most segments of domains I and II for the two proteins.Moreover, this overall arrangement of Cholix relative to PE is even morestriking since these two proteins with similar elements were derivedfrom two distinct directions: P. aeruginosa is a GC-rich bacterium whileV. cholera is AT-rich. That these two toxins evolved from two differentgenetic directions to arrive at nearly the same structure but with only32% amino acid homology suggests that structural and functionalsimilarities of Cholix and PE are likely based upon similar survivalpressures rather than through similar genetic backgrounds. The very lowamino acid homology of domains I and II for these two proteins stressthe functional importance of the folded structures of these two proteinsand not their amino acid sequences.

The C-terminal portion of Cholix and PE appear to function in theintoxication of cells through ADP-ribosylation of EF2 in comparableways. Recent studies where the latter half of Cholix (domain I deleted)targeted to cancer cells through conjugation to an antibody directed tothe transferrin receptor suggests that the C-terminal portions of PE andCholix involved in ADP-ribosylation of EF2 are indeed functionallysimilar; Sarnovsky, R., et al., Cancer Immunol Immunother 59:737-746(2010). While this distal portion of Cholix is 36% identical and 50%similar to PE, polyclonal antisera raised in animals as well as serafrom patients having neutralizing immune responses to this same distalportion of PE failed to cross-react with this latter portion of Cholix.Similarly, antisera raised to this Cholix failed to cross-react with PE.This data suggests that while both PE and Cholix share a capacity tointoxicate cells through a similar mechanism and that these two proteinsshare a common core structure, there are striking differences in theirelements that are expressed at the surface of these proteins.

As previous studies using PE have demonstrated that this toxin readilytransports across polarized monolayers of epithelial cells in vitro andin vivo without intoxication; Mrsny, R. J., et al., Drug Discov Today,7(4): p. 247-58 (2002), the present inventors have commenced research tofurther evaluate the properties and biology of Cholix, with a particularfocus on the functional aspects of the proximal portions of Cholix;specifically, the use of domains I and II to facilitate transport acrossintestinal epithelial monolayers. As domains I and IIa appeared to bethe only essential elements of PE required for epithelial transcytosis,it was particularly important to examine these same domains in Cholix.As stated previously, there is only ˜15%-25% amino acids homology overmost of the regions that would be considered to be part of domains I andIIa. The present inventors examined the domains though a series ofstudies: monitoring the biological distribution of Cholix followingapplication to epithelial surfaces in vivo, assessment of Cholixtranscellular transport characteristics across polarized epithelial cellmonolayers in vitro, and delivery of a biologically-active cargogenetically integrated into the Cholix protein at its C-terminus.Preliminary data generated by genetically fusing the first two domainsof Cholix (amino acids 1-386) to green fluorescent protein (GFP) orchemically coupling these expressed domains to 100 nm diameter latexbeads demonstrated that Cholix attached to 100 nm latex beads wereobserved to transport across intestinal epithelial monolayers in vitroand in vivo. That the GFP cargo retained its fluorescent characterduring and after the transcytosis process also support the contentionthat Cholix utilizes a non-destructive (or privileged) traffickingpathway through polarized epithelial cells. This outcome bodes well forits (repeated) application as a tool to deliver biologically activecargos across epithelial barriers of the body, such as those in therespiratory and gastrointestinal tracts.

Also of important note from the preliminary studies is the observationwhich suggests an apparent cell receptor interaction difference betweenPE and Cholix. As stated previously, PE enters into epithelial cellsafter domain I of PE binds to the membrane protein α2-macroglobulin, aprotein which is also known as the low-density lipoproteinreceptor-related protein 1 (LRP1) or CD91. While the exact identity ofthe surface receptor for Cholix has not been established, preliminarystudies suggest that Cholix does not intoxicate some cell lines thatexpress CD91 but intoxicates some cell lines that do express CD91. It iscurrently unclear what other receptors, beyond CD91, might be involvedepithelial transcytosis of PE. Nevertheless, Cholix and PE appear tohave distinct cell receptor interactions, demonstrating cleardifferences that are sufficient to suggest very different andunanticipated applications for both oral biologics and the intracellulardelivery of bioactive agents.

DISCLOSURE OF INVENTION

The present invention is based on the membrane-trafficking properties ofCholix and the demonstration that Cholix transports efficiently acrosspolarized epithelial cells of the airway and intestine, suggesting thatCholix-derived polypeptide sequences (including the proximal elements ofthe protein) can be used for the efficient transcytosis of protein andnanoparticles, representing a strategy for novel pharmaceuticalapplications.

As such, one aspect of the present invention is to provide isolateddelivery constructs (e.g., genetic fusions or chemical constructs)comprising a transporter domain (e.g., a Cholix-derived polypeptidesequence) and a cargo. Both the transport domain and cargo may beexpressed/linked in varying stoichiometric ratios and spatialorganization. Different cargos may also be expressed/linked on the sameconstruct. In preferred embodiments such cargo may include one, or anyof: proteins, peptides, small molecules, siRNA, PNA, miRNA, DNA,plasmid, and antisense.

Another aspect of the present invention is to provide for the ability todeliver cargo, such as macromolecules, without injections.

Another aspect of the present invention is to provide for the ability todeliver cargo, such as (but not limited to) macromolecules, smallmolecules, siRNA, PNA, miRNA, DNA, plasmid and antisense molecules, intointracellular compartments where their activity is required.

Another aspect of the present invention is to provide for the transportof cargo via delivery of nanoparticles and/or dendrimer-based carriersacross biological membranes.

Methods of administration/delivery contemplated for use in the presentinvention include, e.g., oral administration, pulmonary administration,intranasal administration, buccal administration, sublingualadministration, ocular administration (including, but not limited to,delivery to the vitreous, cornea, conjunctiva, sclera, and posterior andanterior compartments of the eye), topical application, injection(needle or needle-free), intravenous infusion, microneedle application,administration via a drug depot formulation, administration viaintrathecal application, administration via intraperitoneal application,administration via intra-articular application, deliveryintracellularly, delivery across blood brain barrier, delivery acrossblood retina barrier, administered for local delivery and action, and/ordelivered for systemic delivery.

In yet another aspect, the invention provides a pharmaceuticalcomposition comprising the delivery constructs and a pharmaceuticallyacceptable carrier.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 depicts an overview of the strategy used for the C-terminalmodification of ntCholix to facilitate fusion with a cargo, in thisinstance, the cargo being Alexa488 flourescent dye.

FIG. 2 depicts the transport of ntCholix-Alexa488® across polarizedintestinal epithelial cells in vitro. Caco-2 cell monolayers wereexposed to test materials for 4 hr. The percentage of materialtransported was determined by standard curve analysis of fluorescencepresent in the samples and presented as an average (N=4). BSA-Alexa488was used as a control.

MODE(S) FOR CARRYING OUT THE INVENTION

As those in the art will appreciate, the foregoing description describescertain preferred embodiments of the invention in detail, and is thusonly representative and does not depict the actual scope of theinvention. It is also to be understood that the terminology used hereinis for the purpose of describing particular embodiments only, and is notintended to limit the scope of the invention defined by the appendedclaims.

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. As used herein, the following terms havethe meanings ascribed to them unless specified otherwise.

The studies underlying the present invention relate to the use ofCholix-derived polypeptide sequences as the transporter domain to beused to prepare isolated delivery constructs to enhance intestinaldelivery of biologically-active therapeutics. Importantly, the systemsand methods described herein provide for the following: the ability todeliver macromolecule doses without injections; the ability to deliver“cargo” into intracellular compartments where their activity isrequired; and the delivery of nanoparticles and/or dendrimer-basedcarriers across biological membranes, which otherwise would have beenimpeded due to the barrier properties of most such membranes.

Mature Cholix toxin (“Cholix”) is an extremely active monomeric protein(molecular weight 70 kD) secreted by Vibrio cholerae, and which iscomposed of three prominent globular domains (Ia, II, and III) and onesmall subdomain (Ib) that connects domains II and III. The amino acidsequence of mature Cholix is provided in Jorgensen, R. et al., J BiolChem, 283(16):10671-10678 (2008) and references cited therein. TheCholix-derived polypeptide sequences used in the preparation of theisolated delivery constructs of the present invention will be derivedfrom the reported 634 amino acid protein sequence of mature Cholix.

Accordingly, the delivery constructs of the present invention comprise atransporter domain. A “transporter domain” as used herein refers tostructural domains which are capable of performing certain functions,e.g., cell recognition (i.e., comprise a receptor binding domain) andtranscytosis (i.e., comprise a transcytosis domain). Generally, thetransporter domains to be used in the preparation of the deliveryconstructs of the present invention are Cholix-derived polypeptidesequences that have structural domains corresponding to the functionaldomains, e.g., Ia and II, of Cholix.

In addition to the portions of the molecule that correspond to Cholixfunctional domains, the delivery constructs of this invention canfurther comprise a macromolecule for delivery to a biologicalcompartment of a subject. In certain embodiments, the macromolecule isselected from the group of a nucleic acid, a peptide, a polypeptide, aprotein, a polysaccharide, and a lipid. In further embodiments, thepolypeptide is selected from the group consisting of polypeptidehormones, cytokines, chemokines, growth factors, and clotting factorsthat are commonly administered to subjects by injection. The sequencesof all of these macromolecules are well known to those in the art, andattachment of these macromolecules to the delivery constructs is wellwithin the skill of those in the art using standard techniques.

The macromolecule can be introduced into any portion of the deliveryconstruct that does not disrupt a cell-binding or transcytosis activity.The macromolecule is connected to the remainder of the deliveryconstruct through a cleavable linker. “Linker” refers to a molecule thatjoins two other molecules, either covalently, or through ionic, van derWaals or hydrogen bonds, e.g., a nucleic acid molecule that hybridizesto one complementary sequence at the 5′ end and to another complementarysequence at the 3′ end, thus joining two non-complementary sequences. A“cleavable linker” refers to a linker that can be degraded or otherwisesevered to separate the two components connected by the cleavablelinker. Cleavable linkers are generally cleaved by enzymes, typicallypeptidases, proteases, nucleases, lipases, and the like. Cleavablelinkers may also be cleaved by environmental cues, such as, for example,changes in temperature, pH, salt concentration, etc. when there is sucha change in environment following transcytosis of the delivery constructacross a polarized epithelial membrane.

In certain embodiments, the delivery constructs further comprise asecond macromolecule that is selected from the group consisting of anucleic acid, a peptide, a polypeptide, a protein, a lipid, and a smallorganic molecule and a second cleavable linker, wherein cleavage at saidsecond cleavable linker separates said second macromolecule from theremainder of said construct. In certain embodiments, the firstmacromolecule is a first polypeptide and said second macromolecule is asecond polypeptide. In certain embodiments, the first polypeptide andthe second polypeptide associate to form a multimer. In certainembodiments, the multimer is a dimer, tetramer, or octamer. In furtherembodiments, the dimer is an antibody.

In certain embodiments, the macromolecule can be selected to not becleavable by an enzyme present at the basal-lateral membrane of anepithelial cell. For example, the assays described in the examples canbe used to routinely test whether such a cleaving enzyme can cleave themacromolecule to be delivered. If so, the macromolecule can be routinelyaltered to eliminate the offending amino acid sequence recognized by thecleaving enzyme. The altered macromolecule can then be tested to ensurethat it retains activity using methods routine in the art.

In certain embodiments, the first and/or the second cleavable linker iscleavable by an enzyme that exhibits higher activity on thebasal-lateral side of a polarized epithelial cell than it does on theapical side of the polarized epithelial cell. In certain embodiments,the first and/or the second cleavable linker is cleavable by an enzymethat exhibits higher activity in the plasma than it does on the apicalside of a polarized epithelial cell.

In certain embodiments, the cleavable linker can be selected based onthe sequence, in the case of peptide, polypeptide, or proteinmacromolecules for delivery, to avoid the use of cleavable linkers thatcomprise sequences present in the macromolecule to be delivered. Forexample, if the macromolecule comprises AAL, the cleavable linker can beselected to be cleaved by an enzyme that does not recognize thissequence.

In addition to the portions of the molecule that correspond to Cholixfunctional domains, the delivery constructs of this invention canfurther comprise a “cargo” for delivery into intracellular compartmentswhere their activity is required. A “cargo” as used herein includes, butis not limited to: macromolecules, small molecules, siRNA, PNA, miRNA,DNA, plasmid and antisense molecules. Other examples of cargo that canbe delivered according to the present invention include, but are notlimited to, antineoplastic compounds, such as nitrosoureas, e.g.,carmustine, lomustine, semustine, strepzotocin; methylhydrazines, e.g.,procarbazine, dacarbazine; steroid hormones, e.g., glucocorticoids,estrogens, progestins, androgens, tetrahydrodesoxycaricosterone;immunoactive compounds such as immunosuppressives, e.g., pyrimethamine,trimethopterin, penicillamine, cyclosporine, azathioprine; andimmunostimulants, e.g., levamisole, diethyl dithiocarbamate,enkephalins, endorphins; antimicrobial compounds such as antibiotics,e.g., .beta.-lactam, penicillin, cephalosporins, carbapenims andmonobactams, .beta.-lactamase inhibitors, aminoglycosides, macrolides,tetracyclins, spectinomycin; antimalarials, amebicides; antiprotazoals;antifungals, e.g., amphotericin .beta., antivirals, e.g., acyclovir,idoxuridine, ribavirin, trifluridine, vidarbine, gancyclovir;parasiticides; antihalmintics; radiopharmaceutics; gastrointestinaldrugs; hematologic compounds; immunoglobulins; blood clotting proteins,e.g., antihemophilic factor, factor IX complex; anticoagulants, e.g.,dicumarol, heparin Na; fibrolysin inhibitors, e.g., tranexamic acid;cardiovascular drugs; peripheral anti-adrenergic drugs; centrally actingantihypertensive drugs, e.g., methyldopa, methyldopa HCl;antihypertensive direct vasodilators, e.g., diazoxide, hydralazine HCl;drugs affecting renin-angiotensin system; peripheral vasodilators, e.g.,phentolamine; anti-anginal drugs; cardiac glycosides; inodilators, e.g.,amrinone, milrinone, enoximone, fenoximone, imazodan, sulmazole;antidysrhythmics; calcium entry blockers; drugs affecting blood lipids,e.g., ranitidine, bosentan, rezulin; respiratory drugs; sypathomimeticdrugs, e.g., albuterol, bitolterol mesylate, dobutamine HCl, dopamineHCl, ephedrine So, epinephrine, fenfluramine HCl, isoproterenol HCl,methoxamine HCl, norepinephrine bitartrate, phenylephrine HCl, ritodrineHCl; cholinomimetic drugs, e.g., acetylcholine Cl; anticholinesterases,e.g., edrophonium Cl; cholinesterase reactivators; adrenergic blockingdrugs, e.g., acebutolol HCl, atenolol, esmolol HCl, labetalol HCl,metoprolol, nadolol, phentolamine mesylate, propanolol HCl;antimuscarinic drugs, e.g., anisotropine methylbromide, atropineSO.sub.4, clinidium Br, glycopyrrolate, ipratropium Br, scopolamine HBr;neuromuscular blocking drugs; depolarizing drugs, e.g., atracuriumbesylate, hexafluorenium Br, metocurine iodide, succinylcholine Cl,tubocurarine Cl, vecuronium Br; centrally acting muscle relaxants, e.g.,baclofen; neurotransmitters and neurotransmitter agents, e.g.,acetylcholine, adenosine, adenosine triphosphate; amino acidneurotransmitters, e.g., excitatory amino acids, GABA, glycine; biogenicamine neurotransmitters, e.g., dopamine, epinephrine, histamine,norepinephrine, octopamine, serotonin, tyramine; neuropeptides, nitricoxide, K.sup.+channel toxins; antiparkinson drugs, e.g., amaltidine HCl,benztropine mesylate, carbidopa; diuretic drugs, e.g., dichlorphenamide,methazolamide, bendroflumethiazide, polythiazide; antimigraine drugs,e.g, carboprost tromethamine mesylate, methysergide maleate. Thetransporter domains of the delivery constructs of the present inventiongenerally comprise a receptor binding domain. The receptor bindingdomain can be any receptor binding domain known to one of skill in theart without limitation to bind to a cell surface receptor that ispresent on the apical membrane of an epithelial cell. Preferably, thereceptor binding domain binds specifically to the cell surface receptor.The receptor binding domain should bind to the cell surface receptorwith sufficient affinity to allow endocytosis of the delivery construct.

In certain embodiments, the receptor binding domain can comprise apeptide, a polypeptide, a protein, a lipid, a carbohydrate, or a smallorganic molecule, or a combination thereof. Examples of each of thesemolecules that bind to cell surface receptors present on the apicalmembrane of epithelial cells are well known to those of skill in theart. Suitable peptides or polypeptides include, but are not limited to,bacterial toxin receptor binding domains, such as the receptor bindingdomains from PE, cholera toxin, Cholix toxin, botulinum toxin, diptheriatoxin, shiga toxin, shiga-like toxin, etc.; antibodies, includingmonoclonal, polyclonal, and single-chain antibodies, or derivativesthereof, growth factors, such as EGF, IGF-I, IGF-II, IGF-III etc.;cytokines, such as IL-1, IL-2, IL-3, IL-6, etc; chemokines, such asMIP-1a, MIP-1b, MCAF, IL-8, etc.; and other ligands, such as CD4, celladhesion molecules from the immunoglobulin superfamily, integrins,ligands specific for the IgA receptor, etc. The skilled artisan canselect the appropriate receptor binding domain based upon the expressionpattern of the receptor to which the receptor binding domain binds.

The receptor binding domain can be attached to the remainder of thedelivery construct by any method or means known by one of skill in theart to be useful for attaching such molecules, without limitation. Incertain embodiments, the receptor binding domain is expressed togetherwith the remainder of the delivery construct as a fusion protein. Suchembodiments are particularly useful when the receptor binding domain andthe remainder of the construct are formed from peptides or polypeptides.

The transporter domains of the delivery constructs of the presentinvention further comprise a transcytosis domain. The transcytosisdomain can be any transcytosis domain known by one of skill in the artto effect transcytosis of chimeric proteins that have bound to a cellsurface receptor present on the apical membrane of an epithelial cell.In preferred embodiments, the transcytosis domain is domain II ofCholix.

Without intending to be limited to any particular theory or mechanism ofaction, the transcytosis domain is believed to permit the trafficking ofthe delivery construct through a polarized epithelial cell after theconstruct binds to a receptor present on the apical surface of thepolarized epithelial cell. Such trafficking through a polarizedepithelial cell is referred to herein as “transcytosis.” Thistrafficking permits the release of the delivery construct from thebasal-lateral membrane of the polarized epithelial cell.

For the delivery of cargo intended for intracellular activity, thedelivery construct comprises an endocytosis domain to traffic the cargointo the cell, and may also comprise a cleavable linker. This includesthe intracellular delivery of cargo using nanoparticle and/ordendrimer-based carriers targeted to the cell surface receptor bydecorating the carrier surface with one or more copies of theendocytosis domain, with or without the use of linkers.

Without intending to be limited to any particular theory or mechanism ofaction, the endocytosis domain is believed to permit the trafficking ofthe delivery construct into a cell after the construct binds to areceptor present on the surface of the cell. Such trafficking into acell is referred to herein as “endocytosis”. This trafficking permitsthe release of the delivery construct into the relevant intracellularcompartment, including (but not limited to) the nucleus and nuclearenvelope, ribosomal vesicles, endoplasmic reticulum, mitochondria, golgiapparatus, and cytosol.

In certain embodiments of the present invention, identification ofproteases and peptidases that function biological processes that occurat the basolateral surface of these cells will be evaluated. Theseproteases and peptidases would fall into several categories that can bedefined by their substrates: 1) pre-pro-hormones and enzymes that aresecreted from epithelial basolateral surfaces and required trimming fortheir activation, 2) active hormones or enzymes whose activity isneutralized by a cleavage event in order to regulate their activity, and3) systemic enzymes or growth factors whose actions at the basolateralsurface are altered by enzymatic modification. Examples of severalpotential activities that fall into these various categories and whichmight be of useful for the basolateral cleavage of carrier-linker-cargoconstructs include members of the S9B prolyl oligopeptidase subfamily,e.g., FAP and DDP IV, which have been described in the art.

The nucleic acid sequences and polynucleotides of the present inventioncan be prepared by any suitable method including, for example, cloningof appropriate sequences or by direct chemical synthesis by methods suchas the phosphotriester method of Narang, et al., Meth. Enzymol.,68:90-99 (1979); the phosphodiester method of Brown, et al., Meth.Enzymol., 68:109-151 (1979); the diethylphosphoramidite method ofBeaucage, et al., Tetra. Lett., 22:1859-1862 (1981); the solid phasephosphoramidite triester method described by Beaucage & Caruthers,Tetra. Letts., 22(20):1859-1862 (1981), e.g., using an automatedsynthesizer as described in, for example, Needham-VanDevanter, et al.Nucl. Acids Res. 12:6159-6168 (1984); and, the solid support method ofU.S. Pat. No. 4,458,066. Chemical synthesis produces a single strandedoligonucleotide. This may be converted into double stranded DNA byhybridization with a complementary sequence, or by polymerization with aDNA polymerase using the single strand as a template. One of skill wouldrecognize that while chemical synthesis of DNA is limited to sequencesof about 100 bases, longer sequences may be obtained by the ligation ofshorter sequences.

In a preferred embodiment, the nucleic acid sequences of this inventionare prepared by cloning techniques. Examples of appropriate cloning andsequencing techniques, and instructions sufficient to direct persons ofskill through many cloning exercises are found in Sambrook, et al.,MOLECULAR CLONING: A LABORATORY MANUAL (2ND ED.), Vols. 1-3, Cold SpringHarbor Laboratory (1989)), Berger and Kimmel (eds.), GUIDE TO MOLECULARCLONING TECHNIQUES, Academic Press, Inc., San Diego Calif. (1987)), orAusubel, et al. (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, GreenePublishing and Wiley-Interscience, NY (1987). Product information frommanufacturers of biological reagents and experimental equipment alsoprovide useful information. Such manufacturers include the SIGMAchemical company (Saint Louis, Mo.), R&D systems (Minneapolis, Minn.),Pharmacia LKB Biotechnology (Piscataway, N.J.), CLONTECH Laboratories,Inc. (Palo Alto, Calif.), Chem Genes Corp., Aldrich Chemical Company(Milwaukee, Wis.), Glen Research, Inc., GIBCO BRL Life Technologies,Inc. (Gaithersberg, Md.), Fluka Chemica-Biochemika Analytika (FlukaChemie AG, Buchs, Switzerland), Invitrogen, San Diego, Calif., andApplied Biosystems (Foster City, Calif.), as well as many othercommercial sources known to one of skill.

Cells suitable for replicating and for supporting recombinant expressionof protein are well known in the art. Such cells may be transfected ortransduced as appropriate with the particular expression vector andlarge quantities of vector containing cells can be grown for seedinglarge scale fermenters to obtain sufficient quantities of the proteinfor clinical applications. Such cells may include prokaryoticmicroorganisms, such as E. coli; various eukaryotic cells, such asChinese hamster ovary cells (CHO), NSO, 292; Yeast; insect cells; andtransgenic animals and transgenic plants, and the like. Standardtechnologies are known in the art to express foreign genes in thesesystems.

The pharmaceutical compositions of the present invention comprise agenetic fusion or chemical construct of the invention and apharmaceutically acceptable carrier. As used herein, “pharmaceuticallyacceptable carrier” means any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like that are physiologically compatible. Someexamples of pharmaceutically acceptable carriers are water, saline,phosphate buffered saline, dextrose, glycerol, ethanol and the like, aswell as combinations thereof. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Additionalexamples of pharmaceutically acceptable substances are wetting agents orminor amounts of auxiliary substances such as wetting or emulsifyingagents, preservatives or buffers, which enhance the shelf life oreffectiveness of the antibody. Except insofar as any conventionalexcipient, carrier or vehicle is incompatible with the deliveryconstructs of the present invention; its use in the pharmaceuticalpreparations of the invention is contemplated.

In certain embodiments, the pharmaceutical compositions of activecompounds may be prepared with a carrier that will protect thecomposition against rapid release, such as a controlled releaseformulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems (J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978).

In certain embodiments, the delivery constructs of the invention can beorally administered, for example, with an inert diluent or anassimilable edible carrier. The compound (and other ingredients, ifdesired) can also be enclosed in a hard or soft shell gelatin capsule,compressed into tablets, or incorporated directly into the subject'sdiet. For oral therapeutic administration, the delivery constructs canbe incorporated with excipients and used in the form of ingestibletablets, buccal tablets, troches, capsules, elixirs, suspensions,syrups, wafers, and the like. To administer a compound of the inventionby other than parenteral administration, it may be necessary to coat thecompound with, or co-administer the compound with, a material to preventits inactivation.

Generally, a pharmaceutically effective amount of the delivery constructof the invention is administered to a subject. The skilled artisan canreadily determine if the dosage of the delivery construct is sufficientto deliver an effective amount of the macromolecule, as described below.In certain embodiments, between about 1 .mu.g and about 1 g of deliveryconstruct is administered. In other embodiments, between about 10 .mu.gand about 500 mg of delivery construct is administered. In still otherembodiments, between about 10 .mu.g and about 100 mg of deliveryconstruct is administered. In yet other embodiments, between about 10.mu.g and about 1000 .mu.g of delivery construct is administered. Instill other embodiments, between about 10 .mu.g and about 250 .mu.g ofdelivery construct is administered. In yet other embodiments, betweenabout 10 .mu.g and about 100 .mu.g of delivery construct isadministered. Preferably, between about 10 .mu.g and about 50 .mu.g ofdelivery construct is administered.

The delivery constructs of the invention offer several advantages overconventional techniques for local or systemic delivery of macromoleculesto a subject. Foremost among such advantages is the ability to deliverthe macromolecule without using a needle to puncture the skin of thesubject. Many subjects require repeated, regular doses ofmacromolecules. For example, diabetics must inject insulin several timesper day to control blood sugar concentrations. Such subjects' quality oflife would be greatly improved if the delivery of a macromolecule couldbe accomplished without injection, by avoiding pain or potentialcomplications associated therewith.

Furthermore, many embodiments of the delivery constructs can beconstructed and expressed in recombinant systems. Recombinant technologyallows one to make a delivery construct having an insertion sitedesigned for introduction of any suitable macromolecule. Such insertionsites allow the skilled artisan to quickly and easily produce deliveryconstructs for delivery of new macromolecules, should the need to do soarise.

In addition, connection of the macromolecule to the remainder of thedelivery construct with a linker that is cleaved by an enzyme present ata basal-lateral membrane of an epithelial cell allows the macromoleculeto be liberated from the delivery construct and released from theremainder of the delivery construct soon after transcytosis across theepithelial membrane. Such liberation reduces the probability ofinduction of an immune response against the macromolecule. It alsoallows the macromolecule to interact with its target free from theremainder of the delivery construct.

Other advantages of the delivery constructs of the invention will beapparent to those of skill in the art.

Example 1

A plasmid construct was prepared encoding mature Vibrio cholera Cholixand used to express the mature Cholix protein in an E. coli expressionsystem as previously described; see, e.g., Jorgensen, R. et al., J BiolChem, 283(16):10671-10678 (2008). A non-toxic mutant form of the Cholixgene (hereinafter referred to as “ntCholix”) was also prepared bygenetic deletion of a glutamic acid at amino acid position 581 (ΔE581)which is analogous to a deletion (ΔE553) in the PE protein that rendersit non-toxic without significantly altering its conformation; Killeen,K. P. and Collier, R. J., Biochim Biophys Acta, 1138:162-166 (1992).Protein expression was achieved using E. coli DH5α cells (Invitrogen,Carlsbad, Calif.) following transformation by heat-shock (1 min at 42°C.) with the appropriate plasmid. Transformed cells, selected onantibiotic-containing media, were isolated and grown in Luria-Bertanibroth (Difco). Protein expression was induced by addition of 1 mMisopropyl-D-thiogalactopyranoside (IPTG). Two hours following IPTGinduction, cells were harvested by centrifugation at 5,000×g for 10 minat 4° C. Inclusion bodies were isolated following cell lysis andproteins were solubilized in 6 M guanidine HCl and 2 mM EDTA (pH 8.0)plus 65 mM dithiothreitol. Following refolding and purification,proteins were stored at ˜5 ml/ml in PBS (pH 7.4) lacking Ca²⁺ and Mg²⁺at −80° C. All proteins used in these studies were confirmed to beat >90% purity based upon size exclusion chromatography.

The ntCholix form was then modified at its C-terminus to allow directchemical coupling through a free sulfhydryl residue located near theC-terminus of the protein. The strategy for the C-terminal modificationis depicted in FIG. 1. The C-terminal modification included acysteine-constrained loop harboring the consensus cleavage sequence(ENLFQS) for the highly selective protease from the tobacco etch virus(TEV), a second cysteine, and a hexa-histadine (His₆) tag. The secondCys was included to form a disulphide bridge with the Cys ultimatelyused for coupling. Adding the His₆ sequence to the protein simplifiedthe purification and the TEV cleavage sequence provided a mechanism toselectively remove the terminal Cys residue following mild reduction.TEV cleavage and mild reduction with 0.1 mM dithiotheitol followingexpression and isolation of the ntCholix constructs allowed for thedirect chemical coupling of a cargo, Alexa Fluor® 488 fluorescent dye,via a maleimide-based reaction as a generic mechanism of cargoattachment. The resultant construct is referred to herein asntCholix-Alexa488. Following TEV protease cleavage, reduction, and cargocoupling through a maleimide reaction with the free sulfhydryl, removalof the freed C-terminal sequence was achieved by a second Ni²⁺ columnchromatography step.

Example 2

Trans-epithelial transport of ntCholix-Alexa488 was assessed usingCaco-2 monolayers in vitro. First, Caco-2 cells (passage number 25-35)were grown to confluent monolayers as previously described; Rubas, W. etal., Pharm Res, 10:113-118 (1993). Briefly, cells were maintained at 37°C. in DMEM/high growth media enriched with 2 mM L-glutamine, 10% fetalbovine serum, and 100 Units of penicillin/streptomycin in an atmosphereof 5% CO₂ and 90% humidity. Cells were passaged every week at a splitratio of 1:3 in 75 cm² flasks and seeded onto prewetted andcollagen-coated permeable (0.4 μm pore size) polycarbonate (Transwell™)filter supports from Corning Costar (Cambridge, Mass.) at a density of63,000 cells/cm². Growth media was replaced every other day. Confluentmonolayers, determined by the acquisition of significanttrans-epithelial resistance (TEER) determine using an volt-ohm-meter(World Precision Instruments, Sarasota, Fla.), were used 20-26 days postseeding.

Two additional materials were also prepared to be used as controls toassess the in vitro transport of Cholix. As an internal control forfilter damage, tetramethylrhodamine isothiocyanate (TRITC)-labeled 70kDa dextan was obtained from commercial source (Sigma). As a control fornon-specific dye-mediated transport we reacted some of the free amineson the surface of bovine serum albumin (BSA; Sigma) with Alexa488carboxylic acid succinimidyl ester (A488-CASE; Invitrogen). The couplingreaction was carried out for 4 hr at room temperature at neutral pH at amolar ratio of 10:1 A488-CASE:BSA at which point excess glycine wasadded to quench the reaction. The resulting purified product contained˜3 Alexa488 molecules per BSA molecule. Tetramethylrhodamineisothiocyanate (TRITC)-labeled 70 kDa dextan (Sigma) was used as aninternal control for filter damage. Fluorescence measurements were madeusing a BMG labtech FLUOstar Omega instrument set at 540 nm excitationand 610 nm emission for TRITC-Dextran (optimal Ex=547 and Em=572) and480 nm excitation and 520 nm emission for Alexa488 proteins (optimalEx=496 and Em=519).

Trans-epithelial transport flux rates were measured in vitro in theapical (Ap) to basolateral (Bl) and the Bl to Ap directions usingpolarized monolayers of Caco-2 cells to describe mucosal to serosal andserosal to mucosal flux events, respectively. Just prior to initiationof a transport study, the transepithelial resistance (TEER) of eachfilter was measured; monolayers TEER reading of <200 Ω·cm2 were excludedfrom the study. Ap and Bl media was removed from included monolayers andthese surfaces were washed with once with phosphate buffered saline(PBS). One set of monolayers then received an Ap (donor) application of100 μL PBS containing 10 μg ntCholix-A488 and 10 μg TRITC-Dextran or 10μg BSA-A488 and 10 μg TRITC-Dextran. Receiver (Bl) compartments thenreceived 500 μL PBS to set the T₀ for the transport study. Both donorand receiver compartments were sampled after 4 hr of incubation at 37°C. to determine the amount of material transported across the monolayerand the amount retained at the apical surface, respectively.

After 4 hour of exposure we observed ˜5% of the material added to theapical surface of these monolayers to be transported (see FIG. 2). Anyfilters showing levels of 75 kDa TRITC-Dextran in the basal compartmentwere excluded from the analysis. A control protein of BSA-Alexa488failed to show any significant levels in the basal compartment over thissame 4 hr period (see FIG. 2). The averages of transport were5.025±1.13% for Cholix and 0.56±0.33 for BSA (N=4). This dataestablishes that a genetically detoxified form of Cholix can efficientlytransport in vitro across polarized monolayers of a human intestinalcancer cell line, Caco-2.

Example 3

Also prepared and expressed in E. coli. was a variant of Cholixtruncated at amino acid A³⁸⁶ (Cholix³⁸⁶) as well as a genetic ligationof green fluorescent protein (GFP) at the C-terminus of Cholix³⁸⁶(Cholix³⁸⁶GFP). Protein expression was achieved using E. coli DH5α cells(Invitrogen, Carlsbad, Calif.) following transformation by heat-shock (1min at 42° C.) with the appropriate plasmid. Transformed cells, selectedon antibiotic-containing media, were isolated and grown in Luria-Bertanibroth (Difco). Protein expression was induced by addition of 1 mMisopropyl-D-thiogalactopyranoside (IPTG). Two hours following IPTGinduction, cells were harvested by centrifugation at 5,000×g for 10 minat 4° C. Inclusion bodies were isolated following cell lysis andproteins were solubilized in 6 M guanidine HCl and 2 mM EDTA (pH 8.0)plus 65 mM dithiothreitol. Following refolding and purification,proteins were stored at ˜5 ml/ml in PBS (pH 7.4) lacking Ca²⁺ and Mg²⁺at −80° C. Cholix³⁸⁶GFP refolding was monitored by acquisition andretention of the fluorescence signature associated with this fluorescentprotein; Sample et al., Chem Soc Rev, 38(10): p. 2852-64 (2009). Greenfluorescent protein (GFP) was purchased from Upstate (Charlottesville,Va.). All proteins used in these studies were confirmed to be at >90%purity based upon size exclusion chromatography.

Polystyrene beads (10 nm diameter) containing a covalently integratedred fluorescent dye with excitation/emission properties of 468/508 nmand having aldehyde surface functional groups (XPR-582) were obtainedfrom Duke Scientific (Palo Alto, Calif.). One hundred μl of XPR-582beads (at 2% solids) was mixed with approximately 2.5 nmoles GFP orCholix³⁸⁶GFP in a final volume of 200 μl neutral (pH 7.0) phosphatebuffered saline (PBS). After 2 hr of gentle rocking at room temperature,20 μl of a 2 mg/ml solution of bovine serum albumin (BSA; Sigma, St.Louis, Mo.) in PBS was added. Preparations were then dialyzed by threecycles of dilution with PBS and concentration using a 100,000 molecularweight cutoff Microcon filter device from Millipore (Bedford, Mass.).Final preparations of coated beads were at 1% solids.

Example 4

A549 (ATCC CCL-185™), L929 (ATCC CRL-2148™), and Caco-2 (ATCC HTB-37™)cells were maintained in 5% CO₂ at 37° C. in complete media: Dulbecco'smodified Eagle's medium F12 (DMEM F12) supplemented with 10% fetalbovine serum, 2.5 mM glutamine, 100 U of penicillin/ml, and 100 μg ofstreptomycin/ml (Gibco BRL, Grand Island, N.Y.). Cells were fed every 2to 3 days with this media (designated complete medium) and passagedevery 5 to 7 days. For assays, cells were seeded into 24- or 96-wellplates and grown to confluence.

Caco-2 cells were grown as confluent monolayers on collagen-coated0.4-μm pore size polycarbonate membrane transwell supports(Corning-Costar, Cambridge, Mass.) and used 18-25 days after attaining atrans-epithelial electrical resistance (TEER) of >250 Ω·cm2 as measuredusing a chopstick Millicell-ERS® voltmeter (Millipore). Apical tobasolateral (A→B) and basolateral to apical (B→A) transport of Cholix orCholix³⁸⁶GFP across these monolayer was determined by measuring theamount of transported protein 4 hr after a 20 μg application at 37° C.TEER measurements and the extent of 10 kDa fluorescent dextran (measuredusing an HPLC size exclusion protocol) were used to verify monolayerbarrier properties during the course of the study. The extent of Cholixtransport was determined by titration of collected media in thecell-based cytotoxicity assay. Transported Cholix³⁸⁶GFP was measured byenzyme linked immunosorbant assay (ELISA) using anti-GFP antibody forcapture and the polyclonal sera to Cholix for detection.

Transport rates across polarized Caco-2 cells monolayers in vitro werecomparable for Cholix, ntCholix and Cholix³⁸⁶GFP as assess by ELISAformat analysis. In the case of Cholix, polarized Caco-2 cells were notintoxicated by the protein when examined for TUNEL detection ofapoptosis or lactate dehydrogenase (LDH) release. Importantly, Cholixand Cholix-based protein chimeras were found to transport efficientlyfrom the apical to basolateral surface of Caco-2 monolayers but not inthe basolateral to apical direction. These transport rates anddirectionality were comparable to that previously observed for PE testedin this same format. Additionally, we observed that addition rabbitanti-Cholix antisera failed to block the effective transport of Cholixor Cholix-related proteins across Caco-2 monolayers in vitro.

Confocal fluorescence microscopy was used to examine the nature ofCholix³⁸⁶GFP transcytosis across Caco-2 monolayers in vitro. A timecourse study showed Cholix³⁸⁶GFP entering into epithelial cells within 5minutes of its apical application and transporting through cells to thebasolateral region of the cell within 15 minutes. In samples exposed toapical Cholix³⁸⁶GFP for 15 minutes with subsequent removal of excessCholix³⁸⁶GFP from the apical chamber, GFP fluorescence was observed tocontinue in the direction of the basolateral surface of the cell and notback toward the apical surface. This unidirectional movement ofCholix³⁸⁶GFP was confirmed by measuring Cholix³⁸⁶GFP content in theapical and basolateral compartments over this time course. Applicationof Cholix³⁸⁶GFP at the basolateral surface of Caco-2 monolayers did notshow any significant fluorescence entering into the cells, consistentwith transport studies. Western blot analysis of transported Cholix,ntCholix and Cholix³⁸⁶GFP suggested that these proteins transportedwithout major alterations.

In vitro studies also showed that 100 nm diameter fluorescent latexbeads chemically coupled to Cholix³⁸⁶GFP efficiently transported acrossCaco-2 monolayers following an apical application. Latex bead selectionwith a 100 nm diameter provided a material that could readily fit withinthe lumen of a 125 nm diameter endosome derived from a clatherin-coatedpit. Thus, these studies suggest Cholix³⁸⁶GFP-latex beads to movethrough polarized Caco-2 cells by a mechanism consistent with endosomeuptake at the apical cell surface followed by endosome-basedintracellular trafficking. Pre-incubation of Cholix³⁸⁶GFP-coupled 100 nmdiameter fluorescent latex beads with anti-Cholix antisera failed toalter the transport of these beads. A similar amount of GFP chemicallycoupled to 100 nm diameter fluorescent latex beads did not facilitatethe in vitro transport of these particles across Caco-2 monolayers.Confocal fluorescence microscopy studies were consistent withdifferences observed for in transcytosis latex bead coated withCholix³⁸⁶GFP versus GFP.

The result that Cholix is capable of transporting across polarizedepithelial barriers similar to PE is unanticipated. While theirstructures are similar as suggested by crystallographic analysis, theirsurfaces amino acid composition is strikingly different; indeed,alignment methods based upon amino acid similarity would not readilymatch these two proteins. This is important in that the ability of apathogen-derived protein, such as these two virulence factors, tointeract with host cell receptors is presumed to involvesurface-expressed amino acid components. As both of these proteins (withtheir substantially different amino acid sequences) transportefficiently across polarized epithelia, it is highly likely that someother mechanism forms that basis for this transport capacity. It is ourcontention that the structural relationships shared by PE and Cholixforms the basis of the inherent capacity for their efficienttranscytosis. While both PE and Cholix proteins would have the capacityto bind to an apical surface receptor to gain access to endosomalcompartments it is more likely that this interaction and the potentialfor other receptors involved in the intracellular trafficking of theseproteins would be based upon conformational structures rather thanspecific amino acids on the protein surface.

All of the articles and methods disclosed and claimed herein can be madeand executed without undue experimentation in light of the presentdisclosure. While the articles and methods of this invention have beendescribed in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to the articlesand methods without departing from the spirit and scope of theinvention. All such variations and equivalents apparent to those skilledin the art, whether now existing or later developed, are deemed to bewithin the spirit and scope of the invention as defined by the appendedclaims. All patents, patent applications, and publications mentioned inthe specification are indicative of the levels of those of ordinaryskill in the art to which the invention pertains. All patents, patentapplications, and publications are herein incorporated by reference intheir entirety for all purposes and to the same extent as if eachindividual publication was specifically and individually indicated to beincorporated by reference in its entirety for any and all purposes. Theinvention illustratively described herein suitably may be practiced inthe absence of any element(s) not specifically disclosed herein. Thus,for example, in each instance herein any of the terms “comprising”,“consisting essentially of”, and “consisting of” may be replaced witheither of the other two terms. The terms and expressions which have beenemployed are used as terms of description and not of limitation, andthere is no intention that in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional features, modificationand variation of the concepts herein disclosed may be resorted to bythose skilled in the art, and that such modifications and variations areconsidered to be within the scope of this invention as defined by theappended claims.

What is claimed is:
 1. An isolated delivery construct comprising: amodified Cholix toxin consisting of amino acid residues Val¹-Ala³⁸⁶ anda therapeutic cargo coupled to the modified Cholix toxin, the isolateddelivery construct capable of delivering the therapeutic cargo.
 2. Theisolated delivery construct according to claim 1, wherein the deliveringis without injections.
 3. The isolated delivery construct according toany one of claims 1 or 2, further comprising a cleavable linker.
 4. Theisolated delivery construct according to claim 1, wherein thetherapeutic cargo is selected from the group consisting of:macromolecules, small molecules, siRNA, PNA, miRNA, DNA, plasmid andantisense molecules.
 5. A pharmaceutical composition comprising anisolated delivery construct according to claim 1 and a pharmaceuticallyacceptable carrier.
 6. The isolated delivery construct according toclaim 1, wherein the therapeutic cargo is a cytokine.
 7. Thepharmaceutical composition according to claim 5 that is for oraladministration, topical administration, pulmonary administration,intra-nasal administration, buccal administration, sublingualadministration or ocular administration.
 8. The pharmaceuticalcomposition according to claim 7 that is for oral administration.
 9. Thepharmaceutical composition according to claim 5 that is formulated in acapsule or tablet.
 10. The pharmaceutical composition according to claim5, comprising from about 1 μg to about 1 g of the isolated deliveryconstruct.
 11. The pharmaceutical composition according to claim 10,wherein the isolated delivery construct is from about 10 μg to about 100mg.
 12. The pharmaceutical composition according to claim 11, whereinthe isolated delivery construct is from about 10 μg to about 1000 μg.